A constitutive expressed phosphate transporter, OsPht1;1, modulates phosphate uptake and translocation in phosphate-replete rice

A number of phosphate (Pi) starvation- or mycorrhiza-regulated Pi transporters belonging to the Pht1 family have been functionally characterized in several plant species, whereas functions of the Pi transporters that are not regulated by changes in Pi supply are lacking. In this study, we show that rice (Oryza sativa) Pht1;1 (OsPT1), one of the 13 Pht1 Pi transporters in rice, was expressed abundantly and constitutively in various cell types of both roots and shoots. OsPT1 was able to complement the proton-coupled Pi transporter activities in a yeast mutant defective in Pi uptake. Transgenic plants of OsPT1 overexpression lines and RNA interference knockdown lines contained significantly higher and lower phosphorus concentrations, respectively, compared with the wild-type control in Pi-sufficient shoots. These responses of the transgenic plants to Pi supply were further confirmed by the changes in depolarization of root cell membrane potential, root hair occurrence, (33)P uptake rate and transportation, as well as phosphorus accumulation in young leaves at Pi-sufficient levels. Furthermore, OsPT1 expression was strongly enhanced by the mutation of Phosphate Overaccumulator2 (OsPHO2) but not by Phosphate Starvation Response2, indicating that OsPT1 is involved in the OsPHO2-regulated Pi pathway. These results indicate that OsPT1 is a key member of the Pht1 family involved in Pi uptake and translocation in rice under Pi-replete conditions.     

Phosphate transporters OsPHT1;9 and OsPHT1;10 are involved in phosphate uptake in rice

We characterized the function of two rice phosphate (Pi) transporters: OsPHT1;9 (OsPT9) and OsPHT1;10 (OsPT10). OsPT9 and OsPT10 were expressed in the root epidermis, root hairs and lateral roots, with their expression being specifically induced by Pi starvation. In leaves, expression of the two genes was observed in both mesophyll and vasculature. High‐affinity Km values for Pi transport of OsPT9 and OsPT10 were determined by yeast experiments and two‐electrode voltage clamp analysis of anion transport in Xenopus oocytes expressing OsPT9 and OsPT10. Pi uptake and Pi concentrations in transgenic plants harbouring overexpressed OsPT9 and OsPT10 were determined by Pi concentration analysis and ³³P‐labelled Pi uptake rate analysis. Significantly higher Pi uptake rates in transgenic plants compared with wild‐type plants were observed under both high‐Pi and low‐Pi solution culture conditions. Conversely, although no alterations in Pi concentration were found in OsPT9 or OsPT10 knockdown plants, a significant reduction in Pi concentration in both shoots and roots was observed in double‐knockdown plants grown under both high‐ and low‐Pi conditions. Taken together, our results suggest that OsPT9 and OsPT10 redundantly function in Pi uptake.     

Comparative proteome analyses of phosphorus responses in maize (Zea mays L.) roots of wild-type and a low-P-tolerant mutant reveal root characteristics associated with phosphorus efficiency

SUMMARY: Low phosphorus (P) availability is a major limitation for plant growth. To better understand the molecular mechanism of P efficiency in maize, comparative proteome analyses were performed on the roots of the low-P-tolerant mutant 99038 and wild-type Qi-319 grown under P-sufficient (+P) or P-deficient (-P) conditions. Over 10% of proteins detected on two-dimensional electrophoresis (2-DE) gels showed expression that was altered twofold or more between the genotypes under +P or -P conditions. We identified 73 (+P) and 95 (-P) differentially expressed proteins in response to phosphate (Pi) starvation. These proteins were involved in a large number of cellular and metabolic processes, with an obvious functional skew toward carbon metabolism and regulation of cell proliferation. Further analysis of proteome data, physiological measurements and cell morphological observations showed that, compared to the wild-type, the low-P-tolerant mutant could accumulate and secrete more citrate under Pi starvation, which facilitates solubilization of soil Pi and enhances Pi absorption. The proportion of sucrose in the total soluble sugars of the low-P-tolerant mutant was significantly higher, and cell proliferation in root meristem was accelerated. This resulted in better developed roots and more advantageous root morphology for Pi uptake. These results indicate that differences in citrate secretion, sugar metabolism and root-cell proliferation are the main reasons for higher tolerance to low-P conditions in the mutant compared to the wild-type. Thus, the mutant displayed specialized P-efficient root systems with a higher capacity for mobilization of external Pi and increased cell division in the root meristem under Pi starvation.     

RNAi-mediated disruption of squalene synthase improves drought tolerance and yield in rice

About one-third of the world's rice area is in rain-fed lowlands and most are prone to water shortage. The identification of genes imparting tolerance to drought in the model cereal plant, rice, is an attractive strategy to engineer improved drought tolerance not only rice but other cereals as well. It is demonstrated that RNAi-mediated disruption of a rice farnesyltransferase/squalene synthase (SQS) by maize squalene synthase improves drought tolerance at both the vegetative and reproductive stages. Twenty-day-old seedlings of wild type (Nipponbare) and seven independent events of transgenic RNAi lines showed no difference in morphology. When subjected to water stress for a period of 32 d under growth chamber conditions, transgenic positives showed delayed wilting, conserved more soil water, and improved recovery. When five independent events along with wild-type plants were subjected to drought at the reproductive stage under greenhouse conditions, the transgenic plants lost water more slowly compared with the wild type, through reduced stomatal conductance and the retention of high leaf relative water content (RWC). After 28 d of slow progressive soil drying, transgenic plants recovered better and flowered earlier than wild-type plants. The yield of water-stressed transgenic positive plants ranged from 14-39% higher than wild-type plants. When grown in plates with Yoshida's nutrient solution with 1.2% agar, transgenic positives from three independent events showed increased root length and an enhanced number of lateral roots. The RNAi-mediated inactivation produced reduced stomatal conductance and subsequent drought tolerance.     

Overexpression of rice phosphate transporter gene OsPT6 enhances phosphate uptake and accumulation in transgenic rice plants

Background and aims

Whereas the expression patterns and kinetic properties of the rice (Oryza sativa) phosphate transporter gene OsPht1; 6 (OsPT6) are well documented, little is known about the biological functions of this gene. The aim of this study was to investigate the roles of OsPT6 in inorganic phosphate (Pi) acquisition and mobilization, and examine its potential to enhance agricultural production.

Methods

Here, we generated OsPT6 overexpression transgenic plants using Wuyujing 7, a widely cultivated variety of japonica rice, and then treated transgenic lines and wild type with different Pi supply in hydroponic and soil experiments to explore the functions of OsPT6 in rice.

Results

The OsPT6-overexpressing rice lines grew better and accumulated more biomass than wild-type plants, and exhibited significant increases in P accumulation in various tissues, including reproductive tissues under both hydroponic and soil culture conditions. Phosphate-uptake experiment using radiolabeled Pi (³³P) showed that the rate of Pi uptake was 75 % and 73 % greater in transgenic plants grown under Pi-sufficient and -deficient conditions, respectively, than the wild-type controls, and that the shoot/root ratio of ³³P was 104 % and 42 % greater, respectively. In addition, the grain yield per transgenic plant was much higher than that of the wild-type plants under field conditions.

Conclusions

Taken together, our results demonstrate that OsPT6 plays a vital role in Pi acquisition and mobilization in rice and suggest that this gene may be used for genetic engineering rice plants that require less Pi fertilizer.     

LEAF TIP NECROSIS1 plays a pivotal role in the regulation of multiple phosphate starvation responses in rice

Although phosphate (Pi) starvation signaling is well studied in Arabidopsis (Arabidopsis thaliana), it is still largely unknown in rice (Oryza sativa). In this work, a rice leaf tip necrosis1 (ltn1) mutant was identified and characterized. Map-based cloning identified LTN1 as LOC_Os05g48390, the putative ortholog of Arabidopsis PHO2, which plays important roles in Pi starvation signaling. Analysis of transgenic plants harboring a LTN1 promoter::β-glucuronidase construct revealed that LTN1 was preferentially expressed in vascular tissues. The ltn1 mutant exhibited increased Pi uptake and translocation, which led to Pi overaccumulation in shoots. In association with enhanced Pi uptake and transport, some Pi transporters were up-regulated in the ltn1 mutant in the presence of sufficient Pi. Furthermore, the elongation of primary and adventitious roots was enhanced in the ltn1 mutant under Pi starvation, suggesting that LTN1 is involved in Pi-dependent root architecture alteration. Under Pi-sufficient conditions, typical Pi starvation responses such as stimulation of phosphatase and RNase activities, lipid composition alteration, nitrogen assimilation repression, and increased metal uptake were also activated in ltn1. Moreover, analysis of OsmiR399-overexpressing plants showed that LTN1 was down-regulated by OsmiR399. Our results strongly indicate that LTN1 is a crucial Pi starvation signaling component downstream of miR399 involved in the regulation of multiple Pi starvation responses in rice.     

SIZ1 regulation of phosphate starvation-induced root architecture remodeling involves the control of auxin accumulation

Phosphate (Pi) limitation causes plants to modulate the architecture of their root systems to facilitate the acquisition of Pi. Previously, we reported that the Arabidopsis (Arabidopsis thaliana) SUMO E3 ligase SIZ1 regulates root architecture remodeling in response to Pi limitation; namely, the siz1 mutations cause the inhibition of primary root (PR) elongation and the promotion of lateral root (LR) formation. Here, we present evidence that SIZ1 is involved in the negative regulation of auxin patterning to modulate root system architecture in response to Pi starvation. The siz1 mutations caused greater PR growth inhibition and LR development of seedlings in response to Pi limitation. Similar root phenotypes occurred if Pi-deficient wild-type seedlings were supplemented with auxin. N-1-Naphthylphthalamic acid, an inhibitor of auxin efflux activity, reduced the Pi starvation-induced LR root formation of siz1 seedlings to a level equivalent to that seen in the wild type. Monitoring of the auxin-responsive reporter DR5::uidA indicated that auxin accumulates in PR tips at early stages of the Pi starvation response. Subsequently, DR5::uidA expression was observed in the LR primordia, which was associated with LR elongation. The time-sequential patterning of DR5::uidA expression occurred earlier in the roots of siz1 as compared with the wild type. In addition, microarray analysis revealed that several other auxin-responsive genes, including genes involved in cell wall loosening and biosynthesis, were up-regulated in siz1 relative to wild-type seedlings in response to Pi starvation. Together, these results suggest that SIZ1 negatively regulates Pi starvation-induced root architecture remodeling through the control of auxin patterning.     

Nitrate transporter NPF7.3/NRT1.5 plays an essential role in regulating phosphate deficiency responses in Arabidopsis

AtNPF7.3/AtNRT1.5, which is a nitrate transporter that drives root-to-shoot transport of NO₃^?, is also involved in modulating the response to K⁺ deprivation in Arabidopsis by affecting root development and K⁺ transport. However, whether NPF7.3/NRT1.5 functions in regulating plant responses to deficiencies of other nutrients remains unknown. In this study, we found that the expression of AtNPF7.3/AtNRT1.5 was predominant in the roots and was substantially induced by phosphate (Pi) starvation. The atnrt1.5 mutants displayed conspicuously longer primary roots along with a significantly reduced lateral root density under Pi-deficient conditions than did the wild-type plants, and these morphological differences in the roots were eliminated to a certain extent by the ethylene synthesis antagonist Co²⁺. Further analyses revealed that the expression of important Pi starvation-induced genes, which are directly involved in Pi transport, mobilization and distribution, were significantly higher in the atnrt1.5 mutants than that in the wild-type plants under Pi-starvation conditions; therefore, the atnrt1.5 mutants retained higher tissue Pi concentrations. Taken together, our results suggest that NPF7.3/NRT1.5 is an important component in the regulation of phosphate deficiency responses in Arabidopsis.     

Positional Signaling and Expression of ENHANCER OF TRY AND CPC1 Are Tuned to Increase Root Hair Density in Response to Phosphate Deficiency in Arabidopsis thaliana

Phosphate (Pi) deficiency induces a multitude of responses aimed at improving the acquisition of Pi, including an increased density of root hairs. To understand the mechanisms involved in Pi deficiency-induced alterations of the root hair phenotype in Arabidopsis (Arabidopsis thaliana), we analyzed the patterning and length of root epidermal cells under control and Pi-deficient conditions in wild-type plants and in four mutants defective in the expression of master regulators of cell fate, CAPRICE (CPC), ENHANCER OF TRY AND CPC 1 (ETC1), WEREWOLF (WER) and SCRAMBLED (SCM). From this analysis we deduced that the longitudinal cell length of root epidermal cells is dependent on the correct perception of a positional signal (‘cortical bias’) in both control and Pi-deficient plants; mutants defective in the receptor of the signal, SCM, produced short cells characteristic of root hair-forming cells (trichoblasts). Simulating the effect of cortical bias on the time-evolving probability of cell fate supports a scenario in which a compromised positional signal delays the time point at which non-hair cells opt out the default trichoblast pathway, resulting in short, trichoblast-like non-hair cells. Collectively, our data show that Pi-deficient plants increase root hair density by the formation of shorter cells, resulting in a higher frequency of hairs per unit root length, and additional trichoblast cell fate assignment via increased expression of ETC1.     

Transgenic rice tolerant to high temperature with elevated contents of dienoic fatty acids

Transgenic rice plants in which the content of dienoic fatty acids was increased as a result of co-suppression of fatty acid desaturase were more tolerant to high temperatures than untransformed wild-type plants, as judged by growth rate and chlorophyll content. When untransformed wild-type and transgenic rice seedlings were incubated at 35 °C, seedlings of the transgenic rice lines showed approximately 1.6 and 2.1 times the growth of untransformed wild-type seedlings, as assayed by shoot and root mass, respectively. The chlorophyll content of the transgenic leaves after 9 d at 35 °C was also higher than that of wild-type rice. The maximum photochemical efficiency of photosystem 2 was also higher in transgenic plants than in wild-type plants upon high temperature stress.